Epoxyscillirosidine Induced Cytotoxicity and Ultrastructural Changes in a Rat Embryonic Cardiomyocyte (H9c2) Cell Line.

Gezina Ferreira,Christoffel Botha,Hamza Isa,Jan Crafford

doi:10.3390/toxins11050284

Gezina Ferreira, Christoffel Botha + Show 2 more

Open Access

https://doi.org/10.3390/toxins11050284

Copy DOI

Abstract

Moraea pallida Bak. (yellow tulp) poisoning is the most important cardiac glycoside-induced intoxication in ruminants in South Africa. The toxic principle, 1α, 2α-epoxyscillirosidine, is a bufadienolide. To replace the use of sentient animals in toxicity testing, the aim of this study was to evaluate the cytotoxic effects of epoxyscillirosidine on rat embryonic cardiomyocytes (H9c2 cell line). This in vitro cell model can then be used in future toxin neutralization or toxico-therapy studies. Cell viability, evaluated with the methyl blue thiazol tetrazolium (MTT) assay, indicated a hormetic dose/concentration response, characterized by a biphasic low dose stimulation and high dose inhibition. Increased cell membrane permeability and leakage, as expected with necrotic cells, were demonstrated with the lactate dehydrogenase (LDH) assay. The LC50 was 382.68, 132.28 and 289.23 µM for 24, 48, and 72 h respectively. Numerous cytoplasmic vacuoles, karyolysis and damage to the cell membrane, indicative of necrosis, were observed at higher doses. Ultra-structural changes suggested that the cause of H9c2 cell death, subsequent to epoxyscillirosidine exposure, is necrosis, which is consistent with myocardial necrosis observed at necropsy. Based on the toxicity observed, and supported by ultra-structural findings, the H9c2 cell line could be a suitable in vitro model to evaluate epoxyscillirosidine neutralization or other therapeutic interventions in the future.

Highlights

Moraea pallida Bak. poisoning in livestock is the most important of all cardiac glycoside-associated plant poisonings in the Republic of South Africa
Yellow tulp poisoning together with other cardiac glycoside toxicoses accounts for about 33% and 10% mortality, due to plant poisonings in cattle, and small stock, respectively [1]
Cell 2.Viability viability increased when the H9c2 cells were exposed to epoxyscillirosidine at low doses

Summary

Introduction

Moraea pallida Bak. (yellow tulp) poisoning in livestock is the most important of all cardiac glycoside-associated plant poisonings in the Republic of South Africa. (yellow tulp) poisoning in livestock is the most important of all cardiac glycoside-associated plant poisonings in the Republic of South Africa. Acute poisoning with severe cardiac rhythm aberrations occurs, and the mortality rate is high. Yellow tulp poisoning together with other cardiac glycoside toxicoses accounts for about 33% and 10% mortality, due to plant poisonings in cattle, and small stock, respectively [1]. In poisoned animals the respiratory, cardiovascular, gastrointestinal, and nervous systems are involved, and signs may include general apathy, tremors, Toxins 2019, 11, 284; doi:10.3390/toxins11050284 www.mdpi.com/journal/toxins. Toxins 2019, 11, 284 weakness of hindquarters, respiratory distress, and at times bruxism and groaning sounds [2]. Microscopic cardiac lesions are myocardial degeneration and necrosis [3]. The toxic principle is a bufadienolide, 1α, 2α-epoxyscillirosidine [3,4].

Objectives

Methods

Results